Deciphering the Function and Regulation of the B Cell Intrinsic a2,6-Sialylation Network

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Glycosylation is an essential post-translational modification and plays a critical role for the stability and function of many proteins. In the first funding period the focus of project P3 was to study the impact of a2,6 sialic acid residues on mouse B cell development and function during steady state and activation. To study this in detail, we generated an in vivo model for a B cell specific knockout of ST6Gal1, the enzyme that adds terminal a2,6 sialic acid residues to sugar structures present on antibodies and other cell surface receptors. Using this mouse model allowed to make several interesting observations. Firstly, we were able to demonstrate that IgG sialylation occurs within B cells and not post IgG secretion, as suggested more recently. Moreover, by investigating B cell development in these mice, we show that the number of mature B cell subsets including follicular B cells, marginal zone B cells, germinal centre B cells and plasma blasts/plasma cells in the spleen and B2 cells in the peritoneal cavity was strongly reduced. In line with this defect in development of antibody producing B cell subsets, we noted a severe reduction in serum IgG levels. Surprisingly, a strong IgG subclass specific effect of a deletion of ST6Gal1 became evident during the steady-state. Thus, while serum IgG2c was virtually absent, IgG1, IgG2b and IgG3 levels were only slightly affected, suggesting an impact of a2,6-linked sialic acid residues on IgG subclass switching during the steady state. Upon vaccination with T cell-independent and T cell- dependent antigens, however, a reduced but clearly detectable level of IgG1/IgG2c or IgG2c antigen-specific antibody responses could be elicited, respectively. This may suggest, that co-stimulatory signals via toll-like receptors or other co-stimulatory molecules may be able to overcome the defect in IgG2c switching during the steady state. In the second funding period we would like build on the results of the first funding period and study on a B cell subpopulation and single cell level where and when enzymes determining the level of a2,6-linked protein sialyation are expressed, how different signaling pathways critical for B cell activation and class-switching are affected by a lack of a2,6-linked sialic acid residues and how the phenotypes observed in inbred mouse models can be translated to the human immune system via the generation of humanized mice with a knockdown or knockout of ST6Gal1 in human B cells.